ABSTRACT Mutations in the gene encoding PTEN (phosphatase and tensin homolog) are linked to cancers and many neurological disorders with altered brain development. PTEN functions at two primary intracellular locations: the plasma membrane and the nucleus. The function of PTEN at the plasma membrane is well established as a phosphatidylinositol (3,4,5)-trisphosphate phosphatase that suppresses tumorigenic phosphoinositide 3- kinase signaling. In contrast, the functions and mechanisms of PTEN in the nucleus remain largely unknown. Especially, our understanding of the function of nuclear PTEN in brain development is far more limited. To address this critical gap, we have generated a new mouse model in which PTEN levels in the nucleus are significantly decreased using CRISPR/Cas9, without affecting its total expression level, plasma membrane localization, or lipid phosphatase activity. We found that these mice display small neurons and brains but do not experience tumorigenesis. In addition to structural brain defects, these mice exhibited increased anxiety in behavioral tests. These data led to our first hypothesis that nuclear PTEN is crucial for normal development of the brain. Second, using proteomic analyses, we identified a previously uncharacterized tyrosine residue that is both subjected to phosphorylation and critical for the nuclear localization of PTEN. We discovered two protein kinases that induce nuclear recruitment of PTEN in a siRNA screen targeting all of kinases in the human genome and potentially phosphorylate this tyrosine residue. These data led to our second hypothesis that tyrosine phosphorylation controls the localization of PTEN in the nucleus. Here, we will test these two hypotheses in the following aims. In Aim 1, we will determine how nuclear PTEN mediates neuronal and brain development in vivo using our new mouse model. In Aim 2, we will define how tyrosine kinases and phosphorylation target PTEN to the nucleus in both human cell lines and primary mouse neurons. We anticipate that the outcomes of this work will have a significant impact on our understanding of the mechanisms and functions of nuclear PTEN in the physiology and pathophysiology of brain development.